Primer for diagnosis of one or more kinds of cancer

ABSTRACT

This invention relates to primers for diagnosis of one or more kinds of cancer and a diagnostic kit comprising said primers. The primers are made from highly homologous areas of twelve MAGE subtypes and eight GAGE subtypes. The diagnostic kit comprising the said primers can detect six MAGE subtypes and eight GAGE subtypes respectively.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is related to the primers for diagnosis ofone or more kinds of cancers and a diagnostic kit containing the aboveprimers, particularly to common primers that can simultaneously detectsix MAGE sub-types from MAGE 1 to MAGE 6 (MAGE 1-6) or eight GAGEsub-types from GAGE 1 to GAGE 8 (GAGE 1-8) and a diagnostic kitcontaining the above common primers.

[0003] 2. Description of the Prior Art

[0004] The diagnosis of cancers has been accomplished through themedical physical examination, X-ray and CT, histological examination,etc. However, these methods have not been appropriate for discriminationof a cancer among a cancer at its initial stage, a minute cancer, and abenign tumor.

[0005] However, the molecular biological diagnostic methods that havebeen developed recently have contributed greatly to the development ofthe cancer diagnostic area owing to their specificity in the diagnosisof cancers and their high sensitivity The most widely used method amongmany molecular biological diagnostic methods is the polymerase chainreaction (PCR) or the reverse transcriptase-polymerase chain reaction(RT-PCR). The abnormal gene, cancerous antigen gene, etc. of a sampleare amplified and detected in these methods.

[0006] Particularly, RT-PCR is a method of detecting mRNA which isexpressed in a particular gene. It may be used for the diagnosis of acancer by examining the property of expression of cancerous antigengenes. The most important matter in the diagnosis of a cancer in theRT-PCR method is selection of a target gene for cancer diagnosis to bedetected (hereinafter referred to as a “cancer diagnosis marker”).Generally, one cancer diagnosis marker is detected.

[0007] The requirements for a cancer diagnosis marker are that it shouldbe expressed specifically in the cancer and that in a large amount in asmany as possible cancers. MAGE (melanoma antigen gene) and GAGE are twokinds of cancer-associated testis antigens that are expressed in manykinds of cancer tissues but are not expressed in normal tissues exceptfor the testis. It has been clarified that MAGE has been expressed inmany cancers such as the stomach cancer (1, 2), esophagus cancer (3),colon cancer (4), lung cancer (5), breast cancer (6, 7), liver cancer(8), leukemia (9), neuroblastoma (10), ovary cancer (11), etc. in manystudies since it was discovered in the melanoma. And it has beenreported that GAGE has been expressed in the melanoma, sarcoma,small-cell carcinoma, head and neck cancer, bladder cancer, ovarycancer, etc. (11, 12). Therefore, MAGE and GAGE are very extensivelyutilized as cancer diagnostic markers since not only they can detectmany kinds of cancers, not a simple kind of cancer, but also they have ahigh specificity for expression of cancer tissues compared to othercancer-associated antigens such as the carcinoembryonic antigen, etc.Also, MAGE and GAGE have a high homogeneity of genes among sub-types.About 12 kinds of sub-types of MAGE have the homogeneity ranging fromabout 56% to 99%, and 8 kinds of sub-types of GAGE have the homogeneityranging from about 82% to 99%. Therefore, it is possible to increase thecancer diagnosis rate by using such same DNA sequence part as a primersince many sub-types of MAGE or GAGE may be detected simultaneouslyduring RT-PCR.

[0008] Accordingly, common primers having a high cancer diagnostic rateand a high cancer diagnosis specificity through selection of MAGE orGAGE as a marker for cancer diagnosis and simultaneous detection of manysub-types of the marker for cancer diagnosis by RT-PCR are suggested inthe present invention.

SUMMARY OF THE INVENTION

[0009] It is therefore an object of the present invention to providewith the primers for the diagnosis of cancers and a diagnostic kitcontaining such primers through simultaneous amplification of manysub-types of MAGEs or GAGEs and detection of these genes in RT-PCR.

[0010] The common primers are manufactured by comparing the genesequences of many sub-types in order to detect many sub-types of MAGEand GAGE. The primers are manufactured by obtaining the information on12 kinds of MAGE genes and 8 kinds of GAGE genes registered in GenBank,comparing the DNA sequence of each gene, analyzing the DNA homogeneity,and selecting a portion having a high homogeneity. The common primersthus manufactured are shown in Table 1 as follows: <TABLE 1> Cancerdiagnostic Primer marker Type Sequence Sequence No. 1 MAGES 1-6 S5′-GGTCACAAAGGCAGAAATGCT-3′ Sequence No. 2 MAGES 1-6 AS5′-GCCCTTGGACCCCACAGGAACTC-3′ Sequence No. 3 MAGES 1-6 S5′-CTGAAGGAGAAGATCTGCC-3′ Sequence No. 4 MAGES 1-6 AS5′-CTCCAGGTAGTTTTCCTGCAC-3′ Sequence No. 5 MAGES 1-6 S5′-CTGAAGGAGAAGATCTGCCWGTG-3′ Sequence No. 6 MAGES 1-6 AS5′-CCAGCATTTCTGCCTTTGTGA-3′ Sequence No. 7 GAGES 1-8 55′-AGTTGGCGAGGAAGATCGAC-3′ Sequence No. 8 GAGES 1-8 AS5′-CTTCTTTTAACACTGTGATTGC-3′ Sequence No. 9 GAGES 1-8 S5′-AGCCTCCTGAARTGATTGG-3′ Sequence No. 10 GAGES 1-8 AS5′-GCGTTTTCACCTCCTCTGGAT-3′

[0011] In Table 1, S means a sense primer, AS means an anti-senseprimer, W means A or T, and R means A or G.

[0012] Sequence No. ½ is the primer for MAGE, but shows a false positivereaction in PCR. It is suspected that the reason for a false positivereaction is that it is very likely that the genes are amplified from thecontaminated generic DNA since these primers are located in one exon.

[0013] Sequence No. ¾ and Sequence No. ⅚ are the primers for MAGE.Sequence No. 3 and Sequence No. 5 are the primers that are located atthe boundary of two exons. No false positive reaction is shown on PCRand nested PCR using Sequence No. ¾ and Sequence No. ⅚ since SequenceNo. 3 and Sequence No. 5 does not bind to the genoric DNA.

[0014] Sequence No. ⅞ and Sequence No. {fraction (9/10)} are the primersfor GAGE. Sequence No. 7 and Sequence No. 8 or Sequence No. 9 andSequence No. 10 are located at difference exons, and no false positivereaction is shown in the experiments in which Sequence No. ⅞ andSequence No. {fraction (9/10)} are used since a long intron is insertedbetween these exons.

[0015] The cell lines used for the diagnosis of cancers include thehuman stomach cancer cell line (SNU484, SNU638, SNU668), head and neckcancer cell line (AMC-HN-3, AMC-HN-4, AMC-HN-7), cervix cancer cell line(HeLa, Caski), lung cancer cell line (NCI H1703, NCI H522), colon cancercell line (HT29), metastatic prostate gland cancer cell line (LN.CAP),and promyelocytic leukemia (HL60) and osteosarcoma cell line (SaOS2).And for the human body cancer tissues and cells, 12 cases of breastcancer, 27 cases of head and neck cancer, 5 cases of stomach cancer, 3cases of thyroid gland cancer, 3 cases of lymphoma, 1 case of cysticadenoma, 1 case of sarcoma, 1 case of dermatofibrosarcoma, 1 case ofmalignant mixed tumor, 9 cases of urine cells of the patients of bladdercancer, and 15 cases of abdominal cavity cells of the patients ofstomach cancer are used. For the reference benign tumor tissues andcells, 18 cases of benign head and neck tumor, 10 cases of normal whiteblood cells, and 10 case of urine cells of normal people and of patientsof urologic inflammation are used.

[0016] Various cancer tissues are kept at −70° C. until they are used,and are pulverized with a grinder after adding RNAzol B (Tel-Test Inc.,U.S.A.), to the tissues.

[0017] The cultured cancer cell lines are dissolved by adding RNAzol Bafter completely removing the culture medium and washing with thephosphate buffered saline (PBS) solution. The abdominal cavity cells ofstomach cancer are collected after opening the abdominal cavity and aremixed with RNAzol B. The normal white blood cells are dissolvedcompletely by adding RNAzol B after only nucleated cells are collectedfrom the blood. The urine cells are mixed with RNAzol B after the cellsare collected from the urine.

[0018] Separation of RNA from the RNAzol B solution in which varioustissues and cells are dissolved is done according to the manufacturer'sinstructions (Tel-Test., U.S.A.).

[0019] The sputum is used for RT-PCR after it is liquefied by adding theequivalent amount of a stabilizer (Roche Diagnostics, Malinheim,Germany) and mixing thoroughly, and MnRNA is separated by using the mRNAseparation reagent (Roche Diagnostics, Mahnheim, Germany).

[0020] PCR may be used for the diagnosis of cancers by using the commonprimers of the present company. cDNA is produced by the reversetranscriptase (RT) reaction after extracting the total RNA or MRNA fromvarious cancer tissues, sputum, blood, urine, abdominal cavity cells,etc. of the patients. MAGE 1-6 or GAGE 1-8 are amplified by performingPCR using the common primers of the present invention (Sequence No. 1and 2 or Sequence No. 3 and 4 or Sequence No. 7 and 8) for cDNA. Manykinds of cancers may be diagnosed simultaneously through detection ofMAGE 1-6 or GAGE 1-8 DNA bands by electrophoresis of the product of PCRafter performing the secondary PCR (nested PCR) using the common primersof the present invention (Sequence No. 5 and 6 or Sequence No. 9 and 10)for the primary product of RT-PCR.

[0021] In the present invention, RT-PCR is used as a method ofamplification of MAGE 1- or 6GAGE 1-8. However, it is not limited to theapplications described in the present specification, and various methodsof amplification of MAGE 1-6 or GAGE 1-8 by using the common primers ofthe present invention are included in as much as it has variousapplications by varying the reagents, cycles, etc. by the experimenter.

[0022] A cancer diagnostic kit including the common primers of thepresent invention and PCR or RT-PCR reagents may be provided with in thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The foregoing and other objects, aspects and advantages will bebetter understood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

[0024]FIG. 1 shows the affects of processing of MAGE primers and DNaseon the detection of MAGE using PCR or RT-PCR;

[0025]FIG. 2 shows the results of the specificity of detection of MAGEusing the RT-PCR method of Sequence No. ¾ and Sequence No. ⅚ primers;

[0026]FIG. 3 shows the results of measuring each sub-type of MAGE andMAGE 1-6 by using Sequence No. ¾ and Sequence No. ⅚ primers in thecancer cell lines;

[0027]FIG. 4 shows the results of measuring each sub-type of MAGE andMAGE 1-6 by using Sequence No.¾ and Sequence No. ⅚ primers in the cancertissues of the patients of breast cancer;

[0028]FIG. 5 shows the results of measuring MAGE 1-6 by using SequenceNo. ¾ and Sequence No. ⅚ primers in the head and neck cancer and benignhead and neck tumor;

[0029]FIG. 6 shows the results of measuring each sub-type of MAGE andMAGE 1-6 by using Sequence No. ¾ and Sequence No. ⅚ primers in thecancer tissues of the patients of head and neck cancer;

[0030]FIG. 7 shows the results of measuring MAGE 1-6 by using SequenceNo. ¾ and Sequence No. ⅚ primers in other cancer tissues and urinecells;

[0031]FIG. 8 shows the results of measuring GAGE 1-8 by using SequenceNo. ⅞ and Sequence No. {fraction (9/10)} primers in the cancer celllines;

[0032]FIG. 9 shows the results of measuring GAGE 1-8 by using SequenceNo. ⅞ and Sequence No. {fraction (9/10)} primers in the cancer tissuesand urine cells;

[0033]FIG. 10 shows the results of measuring MAGE 1-6 by using SequenceNo. ¾ and Sequence No. ⅚ primers in the lung cancer tissues and sputum;and

[0034]FIG. 11 shows the results of measuring MAGE 1-6 by using SequenceNo. ¾ and Sequence No. ⅚ primers in the blood.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

[0035] The present invention is illustrated in more details belowalthough the content of the present invention is not limited to thefollowings:

[0036] Firstly, the gene expression of MAGE and GAGE and the manufactureof primers are illustrated.

[0037] The DNA sequences are compared and analyzed by using the DNAsisprogram after obtaining the information on 12 kinds of MAGE genes and 8kinds of GAGE genes registered in GenBank. It is shown that thehomogeneity of genes of the MAGE type (cDNA) is 56%-99% and thehomogeneity of genes of the GAGE type (cDNA) is 82%-99%. The portionshaving the same or similar DNA sequence of each sub-type are designed tobe the primers.

[0038] The synthesis is completed after mounting dATP, dTTP, dCTP, anddGTP which are the materials for primer synthesis and the columns forsynthesis to the automatic DNA synthesizer (Expedite TM Nucleic AcidSynthesis System of PerSeptive Biosystem Company) and inputting thesequence to be synthesized. The synthesized primers are used bymeasuring their concentrations with a UV spectrophotometer after theanimonia processing procedure and refining procedure. The primers thusmanufactured are shown in Table 1.

[0039] Sequence No. 1 and Sequence No. 2 (Sequence No. ½) and SequenceNo. 3 and Sequence No. 4 (Sequence No. ¾) are the MAGE primers and areused for RT-PCR or the primary PCR, and Sequence No. 5 and Sequence No.6 (Sequence No. ⅚) are used for the secondary PCR (nested PCR).

[0040] Sequence No. 7 and Sequence No. 8 (Sequence No. ⅞) and SequenceNo. 9 and Sequence No. 10 (Sequence No. {fraction (9/10)}) are theprimers for the detection of GAGEs, where Sequence No. ⅞ is used forRT-PCR or the primary PCR, and Sequence No. {fraction (9/10)} is usedfor the nested PCR.

[0041] Secondly, the procedures for separation of the total RNA aredescribed below:

[0042] Various cancer tissues are kept at −70° C. before they are used.The tissues are pulverized with a grinder after adding 1-2 ml of RNAzolB (Tel-Test Inc., U.S.A.) to the tissues. The cultivated cancer celllines are separated by adding RNAzol B after completely removing theculture medium and washing culture plate once with the PBS. Theabdominal cavity cells of stomach cancer are collected throughcentrifugal separation at 1,200 rpm for 5 minutes after collecting theabdominal cavity cells by adding 100 ml of physiological saline solutionto the lower abdominal cavity after opening the abdominal cavity. Afterthe cells are washed once with the PBS, they are mixed by adding RNAzolB to the cell pellet. In the separation of normal white blood cells,only nucleated cells are collected through centrifugal separation at1,200 rpm for 5 minutes after letting them stand still for 17 secondsafter adding 20 ml of sterilized distilled water (DEPC-DW) to 5 ml ofthe blood containing an anti-coagulant and mixing by adding 20 ml of thetwice concentrated PBS. The cells are dissolved completely by adding 1ml of RNAzol B to the cells collected. The urine cells are collectedthrough centrifugal separation of 150 ml of urine. The cells are washedonce by adding the PBS and are mixed with 1 ml of RNAzol B.

[0043] The protein layer and RNA are separated from each other throughcentrifugal separation at 12,000 rpm for 15 minutes after adding the{fraction (1/10)} portion of chloroform to the RNAzol B solution inwhich various tissues and cells are dissolved and mixing themthoroughly. And the RNA solution of the separated supernatant iscollected carefully and transferred into a 1.5l test tube. RNA isprecipitated by adding 100% isopropanol in the same amount to the RNAsolution, mixing, and keeping at −20° C. for longer than 16 hours. ThenRNA is precipitated through centrifugal separation of the mixedRNA-isopropanol solution at 12,000 rpm, the supernatant is removed, RNAprecipitates are washed by adding 1 ml of 70% cold ethanol to the above,and the upper ethanol solution is removed completely through centrifugalseparation, RNA precipitates are dissolved into the sterilized distilledwater (DEPC-DW), and the concentration and purity of RNA are measured byusing a spectrophotometer. A part of reagents is processed at 37° C. for1 hour by adding 100 μg of RNA to the DNase solution (20-mM MgCl₂, ₂₀-mMtris-HCL, 0.4-U DNase 1, 0.8-U RNasin), and RNA is extracted again andis used for RT-PCR or RNA PCR as described in the above.

[0044] The sputum is completely liquefied by adding a stabilizer (RocheDiagnostics, Mahnheim, Germany) in the same amount and mixingthoroughly, and mRNA is separated by using a separation reagent (RocheDiagnostics, Mahnheim, Germany)) and is used for RT-PCR.

[0045] Thirdly, the total RNA is collected and PCR is performed asfollows in order to see if the genomic MAGE DNA is contaminated. In thefirst step, the PCR reaction solution is made by mixing 3 μl of the 10XPCR buffer solution, 1.8 μl of 250-mM MgCl₂, 0.3 μl of 10-mM dATP, 0.3 lof 10-mM dGTP, 0.3 μl of 10-mM dTTP, 0.3 μl of 10-mM dCTP, 0.25 μl of50-μM sense and anti-sense primers, and 0.25 μl of the Taq polymerase (5U/μl, Promega Co., U.S.A.) to make up 25 μl of the final PCR reactionsolution. The PCR reaction solution is put into a PCR tube, 5 μl of thetotal RNA solution (0.1 μg/μl) is added to the above solution and mixed,1 drop of a mineral oil is dropped, and the entire solution is put intoa PCR machine (Cetus 480, Perkin Elmer Co., U.S.A.) in order to performPCR under the following conditions: Firstly, DNA is denatured by heatingthe above solution at 94° C. for 5 minutes and reacting for 30-35 cyclesin which 1 cycle is for 30 seconds at 94° C., 45 seconds at 57° C., and45 seconds at 72° C., and PCR is completed by treating the abovesolution at 72° C. for 5 minutes finally. Then the amplified DNA bandsare observed by using a UV transilluminator after adding the product ofPCR to 1% agarose gel and electrophoresis.

[0046] Fourthly, the reverse transcriptase-polymerase chain reaction(RT-PCR) is performed. RNA is denatured by keeping the total RNAsolution in a 70° C. water bath for 10 minutes and kept in ice. Firstly,2 μl of the 5X RT buffer solution, 0.25 μl of 10-mM dATP, 0.25 μl of10-mM dGTP, 0.25 μl of 10-mM dTTP, 0.25 μl of 10- mM dCTP, 0.25 μl ofMMLV reverse transcriptase (200U/μl ), 0.25 μl of RNase inhibitor (28U/μl ), 0.5 μl of 50μM oligo dT primer, and 4 μl of the sterilizeddistilled water (DEPC-DW) are put into a PCR tube in order to make an RTreaction solution. To the RT reaction solution, 2 μl of the total RNAsolution (1 μg/μl) that is kept in ice is added and 1 drop of a mineraloil is dropped, and the entire solution is kept at a room temperaturefor 10 minutes. The reverse transcriptase reaction is completed byputting this test tube into the PCR machine and treating with heat at42° C. for 60 minutes. And the product of reverse transcriptase reactionis diluted with distilled water at the ratio of 1:1 and is used for PCR.PCR is performed under the following conditions by adding 5 μl of theproduct of reverse transcriptase reaction into 25 μl of the PCR reactionsolution as described in the above, mixing them, dropping 1 drop of amineral oil, and putting the solution into the PCR machine: Firstly, DNAis denatured by heating the solution at 94° C. for 5 minutes andreacting for 18-35 cycles in which 1 cycle is for 30 seconds at 94° C.,45 seconds at 57° C., and 45 seconds at 72° C., and PCR is completed byprocessing the above at 72° C. for 5 minutes finally.

[0047] Fifthly, in the nested PCR, the product of PCR or RT-PCR reactionis diluted 10 times with distilled water, its 5-μl portion is taken, andthe secondary PCR is performed after adding PCR reagents as described inthe above.

[0048] Sixthly, the affects of processing of the MAGE primer and DNaseon the detection of MAGE are reviewed.

[0049] The total RNA is extracted from the cultured stomach cancer cellline (SNU484, SNU638, SNU668), head and neck cancer cell line (AMC-HN-3,AMC-HN-4, AMC-HN-7), cervix cancer cell line (HeLa, Caski), lung cancercell line (NCI H1703, NCI H522), colon cancer cell line (HT29),metastatic prostate gland cancer cell line (LN.CAP), and promyelocyticleukemia (HL60) and osteosarcoma cell line (SaOS2), after which PCR andRT-PCR for detecting MAGE are performed. (Refer to FIG. 1.)

[0050] A in FIG. 1 is the result of PCR of the total RNA by usingSequence No. {fraction (1/2,)} and B is the result of PCR of the totalRNA by using Sequence No. ½ after processing DNase to the total RNA. Cis the result of PCR of the total RNA by using Sequence No. {fraction(3/4,)} and D is the result of PCR of the total RNA by using SequenceNo. ¾ and further of nested PCR by using Sequence No. {fraction (5/6.)}In conclusion, it is necessary to perform DNase processing in order touse Sequence No. {fraction (1/2,)} but MAGE may be detected withoutDNase processing by Sequence No. ¾ and Sequence No. {fraction (5/6,)}and no false positive reaction by the amplification of genomic DNA isshown. And Sequence No. ¾ and Sequence No. ⅚ may detect at least one of6 kinds of sub-types of MAGE (MAGE 1-6) from MAGE 1 to MAGE 6.

[0051] Seventhly, the cancer diagnosis specificity of the RT-PCR methodby using Sequence No. ¾ and Sequence No. ⅚ is evaluated. In order tostudy the specificity of the RT-PCR method by using Sequence No. ¾ andSequence No. {fraction (5/6,)} the SNU484 cell line and normal whiteblood cells are extracted to perform RT-PCR and nested PCR. The resultsshow that MAGE 1-6 are detected only in RT-PCR and nested PCR in SNU484,and generic DNA is not amplified when PCR and nested PCR are performed.(Refer to FIG. 2A.) Further, no MAGE 1-6 is detected in normal whiteblood cells. (Refer to FIG. 2B.)

[0052] Eighthly, in the measurement of MAGEs in the cancer cell line,each sub-type of MAGE and MAGE 1-6 are measured for 14 kinds of cancercell lines. Each sub-type of MAGE is detected by RT-PCR using SequenceNo. 3 and MAGE sub-type specific primers. MAGE 1-6 is detected by RT-PCRusing Sequence No. ¾ and nested PCR using Sequence No. {fraction (5/6.)}The experiments for measuring MAGE 1-6 show a higher positive rate(78.6%) compared to that in the measurement of each sub-type. (Refer toFIG. 3.)

[0053] Ninthly, in the measurement of MAGEs in the breast cancer, eachsub-type of MAGE and MAGE 1-6 are measured for the cancer tissues of thepatients of breast cancer. RT-PCR having Sequence No. ¾ as primer andnested PCR using the Sequence No. 5 primer and MAGE sub-type specificprimer are performed, but RT-PCR using Sequence No. ¾ and nested PCRusing Sequence No. ⅚ are performed for the detection of MAGE 1-6. Ahigher positive rate (91.2%) is shown in the measurement of MAGE 1-6compared to that in the measurement of each sub-type. (Refer to FIG. 4.)

[0054] Tenthly, in the measurement of MAGEs in the head and neck cancerand benign head and neck tumor, MAGE 1-6 are measured. MAGE 1-6 ismeasured by RT-PCR using Sequence No. ¾ and nested PCR using SequenceNo. {fraction (5/6.)} Among 27 cases of cancers, 19 cases are positive(70.3%), and none is detected in the benign tumor. (Refer to FIG. 5,where M means a size marker and—is PCR without adding CDNA.)

[0055] Eleventhly, in the measurement of MAGE sub-types in the head andneck cancers, each sub-type of MAGE and MAGE 1-6 are measured in thesamples in which MAGE 1-6 are detected among the samples of head andneck cancer. RT-PCR using Sequence No. ¾ as a primer and nested PCRusing the Sequence No. 5 and MAGE sub-type specific primers areperformed, and MAGE 1-6 are measured in terms of RT-PCR and nested PCRby using Sequence No. ¾ and Sequence No. {fraction (5/6,)} respectively.The method of measurement of each sub-type has a lower efficiency thanthat of the method of detection of MAGE 1-6. (Refer to FIG. 6.)

[0056] Twefthly, the measurement of MAGE 1-6 in other cancer tissues andabdominal cavity and urine cells is reviewed.

[0057] MAGE 1-6 are measured in terms of RT-PCR and nested PCR by usingSequence No. ¾ and Sequence No. {fraction (5/6,)} respectively. Theobjects of measurement include the thyroid gland cancer (A), lymphoma(B), cystic adenoma (C), sarcoma (D), dermatofibrosarcoma (E), malignantmixed tumor (F), abdominal cavity cell of the patients of stomach cancer(G), urine of the patients of bladder cancer (H), and urine of thepatients of other diseases (I). (Refer to FIG. 7, where M means a sizemarker, and—is PCR without adding cDNA.)

[0058] No MAGE 1-6 are detected in the thyroid gland cancer, but variouskinds of MAGEs are detected in the abdominal cells of the patients ofremaining cancers and stomach cancer and the urine cells of the patientsof bladder cancer.

[0059] Thirteenthly, in the measurement of GAGEs in cancer tissues andurine cells, GAGE 1-8 of 14 kinds of cancer cell lines are measured.GAGE 1-8 are measured in terms of RT-PCR or PCR using Sequence No. ⅞ ornested PCR using Sequence No. {fraction (9/10. )}

[0060] No GAGE is detected in case of PCR and nested PCR without thereverse transcriptase reaction, and positive reactions are shown in 13reactions axnong 14 reactions (92.9%) in case of PCR and nested PCRafter the reverse transcriptase reaction. (Refer to FIG. 8.) In thestomach cancer, 80% of reactions are shown to be positive to GAGEs,85.7% is shown to be positive in breast cancer, and 55.6% is shown to bepositive in the urine cells of bladder cancer. And no positive reactionsare shown in the urine of the patients of diseases other than thecancer. (Refer to FIG. 9.)

[0061] Fourteenthly, in the measurement of MAGEs in the lung cancertissues and sputum, MAGE 1-6 are measured in 8 cases of lung cancertissues, 14 cases of sputum of the patients of lung cancer, and 16 casesof sputum of hospitalized patients of diseases other than the lungcancer. They are measured in terms of RT-PCR and nested PCR by usingSequence No. ¾ and Sequence No. {fraction (5/6,)} respectively. MAGE 1-6are detected in 7 cases among 8 cases of lung cancer tissues, and 4cases among 14 cases of sputum of the patients of lung cancer. And noMAGEs are detected at all in 16 cases of sputum of hospitalized patientsof diseases other than the lung cancer. (Refer to FIG. 10.)

[0062] Fifteenthly, in the measurement of MAGEs in the blood, MAGEs aremeasured in 20 cases of the blood of patients of cancer and 15 cases ofthe blood of normal people. They are measured in terms of RT-PCR andnested PCR by using Sequence No. ¾ and Sequence No. {fraction (5/6,)}respectively. MAGE 1-6 are detected in 3 cases among 20 cases of theblood of the patients of cancer, and none is detected in the blood ofnormal people. (Refer to FIG. 11, where + is a sample in which 5 cellsof SNU484 (MAGEs 1-6 benign cells) to 5 ml of the blood of normalpeople.)

[0063] Primer Sequence ¾ an Sequence No. ⅚ that detect MAGE 1 to MAGE 6simultaneously and Sequence No. ⅞ and Sequence No. {fraction (9/10)}that detect GAGE 1 to GAGE 8 simultaneously may be used as cancerdiagnostic kits having a high specificity and a high diagnosis rate ofcancer compared to the method of measuring each sub-type.

[0064] Also, they not only have a very high specificity for thediagnosis of cancers since none is shown to be positive in variousnormal cells and benign tumors, but also can detect MAGEs in urine cellsor sputum cells by using these common primers. Therefore, they may beused for cancer diagnostic kits for non-hygroscopic samples, as well astissues. The present invention is also advantageous in that the time andexpenses may be reduced through simultaneous measuring of many sub-typesof MAGEs or GAGEs.

References

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1 10 1 21 DNA Artificial Sequence primer targeting MAGE 1-6; senseprimer type 1 ggtcacaaag gcagaaatgc t 21 2 23 DNA Artificial Sequenceprimer targeting MAGE 1-6; antisense primer type 2 gcccttggac cccacaggaactc 23 3 19 DNA Artificial Sequence primer targeting MAGE 1-6; senseprimer type 3 ctgaaggaga agatctgcc 19 4 21 DNA Artificial Sequenceprimer targeting MAGE 1-6; antisense primer type 4 ctccaggtag ttttcctgcac 21 5 23 DNA Artificial Sequence primer targeting MAGE 1-6; senseprimer type 5 ctgaaggaga agatctgccw gtg 23 6 21 DNA Artificial Sequenceprimer targeting MAGE 1-6; antisense primer type 6 ccagcatttc tgcctttgtga 21 7 20 DNA Artificial Sequence primer targeting GAGE 1-6; senseprimer type 7 agttggcgag gaagatcgac 20 8 22 DNA Artificial Sequenceprimer targeting GAGE 1-6; antisense primer type 8 cttcttttaa cactgtgattgc 22 9 19 DNA Artificial Sequence primer targeting GAGE 1-6; senseprimer type 9 agcctcctga artgattgg 19 10 21 DNA Artificial Sequenceprimer targeting GAGE 1-6; antisense primer type 10 gcgttttcacctcctctgga t 21

What is claimed is:
 1. A primer selected from Sequence No. 3 throughSequence No.
 10. 2. A common primer comprised of two sequences selectedfrom Sequence No. 3 through Sequence No.
 10. 3. The common primer inclaim 2, wherein said common primer is selected from Sequence No. 3 andSequence No. 4, Sequence No. 5 and Sequence No. 6, Sequence No. 7 andSequence No. 8, and Sequence No. 9 and Sequence No.
 10. 4. A cancerdiagnostic kit, wherein the main component of said cancer diagnostic kitis a common primer selected in claim 1 through claim
 3. 5. A method ofdiagnosis of cancers by using a primer selected from Sequence No. 3through Sequence No.
 10. 6. A method of diagnosis of cancers by using acommon primer comprised of two sequences selected from Sequence No. 3through Sequence No.
 10. 7. The method of diagnosis of a cancer in claim6, wherein said common primer is selected from Sequence No. 3 andSequence No. 4, Sequence No. 5 and Sequence No. 6, Sequence no. 7 andSequence No. 8, and Sequence No. 9 and Sequence No.
 10. 8. The method ofdiagnosis of cancers in any of claim 5 through claim 7, wherein one ormore kinds of cancers are diagnosed simultaneously.
 9. The method ofdiagnosis of cancers in any of claim 5 through claim 8, wherein PCR isperformed for said diagnosis.
 10. The method of diagnosis of cancers inclaim 9, wherein the secondary PCR of RT-PCR and nested PCR is performedfor said diagnosis.
 11. A cancer diagnostic kit containing a primerselected from Sequence No. 3 through Sequence No. 10 and PCR reagents.12. A cancer diagnostic kit containing a common primer comprised of twosequences selected from Sequence No. 3 through Sequence No. 10 and PCRreagents.
 13. The cancer diagnostic kit in claim 12, wherein a commonprimer selected from Sequence No. 3 and Sequence No. 4, Sequence No. 5and Sequence No. 6, Sequence No. 7 and Sequence No. 8, and Sequence No.9 and Sequence No. 10 and PCR reagents are contained in said cancerdiagnostic kit.